Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During
infection, HCV NS5A
protein activates PI4KIIIα
enzyme, causing massive production and redistribution of
phosphatidylinositol 4-phosphate (PI4P)
lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for
doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-
phosphate adaptor
protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV
RNA synthesis. Indeed, FAPP2 has two functional
lipid-binding domains specific for PI4P and
glycosphingolipids. While expression of the PI4P-binding
mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the
glycosphingolipid-binding
mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some
glycosphingolipids, whereas adding these
lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of
glycosphingolipids in HCV
RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or
double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase
proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and
glycosphingolipid transport to the HCV RC.
IMPORTANCE: Like most viruses with a positive-sense
RNA genome, HCV replicates its
RNA on remodeled host membranes composed of
lipids hijacked from various internal membrane compartments. During
infection, HCV induces massive production and retargeting of the PI4P
lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and
glycosphingolipid-
binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of
glycosphingolipids in the HCV life cycle and suggests a link between PI4P and
glycosphingolipids in HCV genome replication.